The pygidial gland secretion of the rove beetle genera Stenus Latreille and Dianous Leach is composed of pyridine and piperidine-derived alkaloids and several terpene compounds. Two-choice bioassays with ants and fish, as well as agar diffusion assays, revealed that the secretion compounds are capable of deterring predators and protecting the beetles from infestation with microorganisms. In addition, the beetles use the secretion for rapid movements on the water surface, a process called skimming. Thus, originally developed to chemically defend the sensitive unprotected abdomen from predator attacks, the secretion of recent Steninae can be designated as multifunctional. Four of the alkaloid compounds occur as different configurational isomers in the secretion. Two-choice tests showed that ants discriminate between stereoisomers of stenusine, while there was no effect visible on bacteria in agar diffusion assays. Furthermore, there are evolutionary trends within the Steninae concerning the secretion composition, as some of the alkaloids primarily occur in phylogenetically basal species, while others are mainly restricted to derived species.